Methods for dynamic vector assembly of DNA cloning vector plasmids

ABSTRACT

A method for using cloning vector plasmids to produce DNA molecules, such as transgenes, in a single cloning step. The transgenes can be used for the purpose of gene expression or analysis of gene expression. The plasmid cloning vectors are engineered to minimize the amount of manipulation of DNA fragment components by the end user of the vectors and the methods for their use. Transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

BACKGROUND OF THE INVENTION

The present invention relates in general to the field of cloning vectorplasmids, and in particular to methods for rapidly assembling DNAconstructs or transgenes with cloning vector plasmids.

The foundation of molecular biology is recombinant DNA technology, whichcan here be summarized as the modification and propagation of nucleicacids for the purpose of studying the structure and function of thenucleic acids and their protein products.

Individual genes, gene regulatory regions, subsets of genes, and indeedentire chromosomes in which they are contained, are all comprised ofdouble-stranded anti-parallel sequences of the nucleotides adenine,thymine, guanine and cytosine, identified conventionally by the initialsA, T, G, and C, respectively. These DNA sequences, as well as cDNAsequences, which are double stranded DNA copies derived from mRNA(messenger RNA) molecules, can be cleaved into distinct fragments,isolated, and inserted into a vector such as a bacterial plasmid tostudy the gene products. A plasmid is an extra-chromosomal piece of DNAthat was originally derived from bacteria, and can be manipulated andreintroduced into a host bacterium for the purpose of study orproduction of a gene product. The DNA of a plasmid is similar to allchromosomal DNA, in that it is composed of the same A, T, G, and Cnucleotides encoding genes and gene regulatory regions, however, it is arelatively small molecule comprised of less than approximately 30,000base-pairs, or 30 kilobases (kb). In addition, the nucleotide base pairsof a double-stranded plasmid form a continuous circular molecule, alsodistinguishing plasmid DNA from that of chromosomal DNA.

Plasmids enhance the rapid exchange of genetic material betweenbacterial organisms and allow rapid adaptation to changes inenvironment, such as temperature, food supply, or other challenges. Anyplasmid acquired must express a gene or genes that contribute to thesurvival of the host or else it will be destroyed or discarded by theorganism, since the maintenance of unnecessary plasmids would be awasteful use of resources. A clonal population of cells containsidentical genetic material, including any plasmids it might harbor. Useof a cloning vector plasmid with a DNA insert in such a clonalpopulation of host cells will amplify the amount of the DNA of interestavailable. The DNA so cloned may then be isolated and recovered forsubsequent manipulation in the steps required for building a DNAconstruct. Thus, it can be appreciated that cloning vector plasmids areuseful tools in the study of gene function, providing the ability torapidly produce large amounts of the DNA insert of interest.

While some elements found in plasmids are naturally occurring, othershave been engineered to enhance the usefulness of plasmids as DNAvectors. These include antibiotic- or chemical-resistance genes and amultiple cloning site (MCS), among others. Each of these elements has arole in the present invention, as well as in the prior art. Descriptionof the role each element plays will highlight the limitations of theprior art and demonstrate the utility of the present invention.

A particularly useful plasmid-born gene that can be acquired by a hostis one that would confer antibiotic resistance. In the daily practice ofrecombinant DNA technology, antibiotic resistance genes are exploited aspositive or negative selection elements to preferentially enhance theculture and amplification of the desired plasmid over that of otherplasmids.

In order to be maintained by a host bacterium, a plasmid must alsocontain a segment of sequences that direct the host to duplicate theplasmid. Sequences known as the origin of replication (ORI) elementdirect the host to use its cellular enzymes to make copies of theplasmid. When such a bacterium divides, the daughter cells will eachretain a copy or copies of any such plasmid. Certain strains of E. colibacteria have been derived to maximize this duplication, producingupwards of 300 copies per bacterium. In this manner, the cultivation ofa desired plasmid can be enhanced.

Another essential element in any cloning vector is a location forinsertion of the genetic materials of interest. This is a syntheticelement that has been engineered into “wild type” plasmids, thusconferring utility as a cloning vector. Any typicalcommercially-available cloning vector plasmid contains at least one suchregion, known as a multiple cloning site (MCS). A MCS typicallycomprises nucleotide sequences that are cleaved by a single endonucleaseenzyme, or a series of endonuclease enzymes, each of which has adistinct recognition sequence and cleavage pattern. The so-calledrecognition sequences of a restriction endonuclease (RE) site encoded inthe DNA molecule comprise double-stranded palindromic sequences. Forsome RE enzymes, as few as 4-6 nucleotides are sufficient to provide arecognition site, while some RE enzymes require a sequence of 8 or morenucleotides. The RE enzyme EcoR1, for example, recognizes thedouble-stranded hexanucleotide sequence: ^(5′)G-A-A-T-T-C^(3′), wherein5′ indicates the end of the molecule known by convention as the“upstream” end, and 3′ likewise indicates the “downstream” end. Thecomplementary strand of the recognition sequence would be itsanti-parallel strand, ^(3′) G-A-A-T-T-C-^(5′). Since every endonucleasesite is a double-stranded sequence of nucleotides, a recognition site of6 nucleotides is, in fact, 6 base pairs (bp). Thus the double strandedrecognition site can be represented within the larger double-strandedmolecule in which it occurs as:

^(5′) . . . G-A-A-T-T-C . . . ^(3′) ^(3′) . . . C-T-T-A-A-G . . . ^(5′).

Like many other RE enzymes, EcoR1 does not cleave exactly at the axis ofdyad symmetry, but at positions four nucleotides apart in the two DNAstrands between the nucleotides indicated by a “/”:

^(5′) . . . G/A-A-T-T-C . . . ^(3′) ^(3′) . . . C-T-T-A-A/G . . . ^(5′),

such that double-stranded DNA molecule is cleaved and has the resultantconfiguration of nucleotides at the newly formed “ends”:

^(5′) . . . G ^(3′) ^(5′) A-A-T-T-C . . . ^(3′)^(3′) . . . C-T-T-A-A ^(5′) ^(3′) G . . . ^(5′)

This staggered cleavage yields fragments of DNA with protruding 5′termini. Because A-T and G-C pairs are spontaneously formed when inproximity with each other, protruding ends such as these are calledcohesive or sticky ends. Any one of these termini can form hydrogenbonds with any other complementary termini cleaved with the samerestriction enzyme. Since any DNA that contains a specific recognitionsequence will be cut in the same manner as any other DNA containing thesame sequence, those cleaved ends will be complementary. Therefore, theends of any DNA molecules cut with the same RE enzyme “match” each otherin the way adjacent pieces of a jigsaw puzzle “match”, and can beenzymatically linked together. It is this property that permits theformation of recombinant DNA molecules, and allows the introduction offoreign DNA fragments into bacterial plasmids, or into any other DNAmolecule.

A further general principle to consider when building recombinant DNAmolecules is that all endonuclease sites occurring within a moleculewill be cut with a particular RE enzyme, not just the site of interest.The larger a DNA molecule, the more likely it is that any endonucleasesite will reoccur. Assuming that any endonuclease sites are distributedrandomly along a DNA molecule, a tetranucleotide site will occur, on theaverage, once every 4⁴ (i.e., 256) nucleotides or bp, whereas ahexanucleotide site will occur once every 4⁶ (i.e., 4096) nucleotides orbp, and octanucleotide sites will occur once every 4⁸ (i.e., 114,688)nucleotides or bp. Thus, it can be readily appreciated that shorterrecognition sequences will occur frequently, while longer ones willoccur rarely. When planning the construction of a transgene or otherrecombinant DNA molecule, this is a vital issue, since such a projectfrequently requires the assembly of several pieces of DNA of varyingsizes. The larger these pieces are, the more likely that the sites onewishes to use occur in several pieces of the DNA components, makingmanipulation difficult at best.

Frequently-occurring endonuclease enzyme sites are herein referred to ascommon sites, and the endonucleases that cleave these sites are referredto as common endonuclease enzymes. Restriction enzymes with cognaterestriction sites greater than 6 bp are referred to as rare restrictionenzymes, and their cognate restriction sites as rare restriction sites.However, there are some endonuclease sites of 6 bp that occur moreinfrequently than would be statistically predicted, and these sites andthe endonucleases that cleave them are also referred to as rare. Thus,the designations “rare” and common” do not refer to the relativeabundance or availability of any particular restriction enzyme, butrather to the frequency of occurrence of the sequence of nucleotidesthat make up its cognate recognition site within any DNA molecule orisolated fragment of a DNA molecule, or any gene or its DNA sequence.

A second class of endonuclease enzymes has recently been isolated,called homing endonuclease (HE) enzymes. HE enzymes have large,non-palindromic asymmetric recognition sites (12-40 base pairs). HErecognition sites are extremely rare. For example, the HE known asI-SceI has an 18 bp recognition site, (5′. . . TAGGGATAACAGGGTAAT . ..3′), predicted to occur only once in every 7×10¹⁰ bp of randomsequence. This rate of occurrence is equivalent to only one site in 20mammalian-sized genomes. The rare nature of HE recognition sites greatlyincreases the likelihood that a genetic engineer can cut a finaltransgene product without disrupting the integrity of the transgene ifHE recognition sites were included in appropriate locations in a cloningvector plasmid.

Since a DNA molecule from any source organism will be cut in identicalfashion by an endonuclease enzyme, foreign pieces of DNA from anyspecies can be cut with an endonuclease enzyme, inserted into abacterial plasmid vector that was cleaved with the same endonucleaseenzyme, and amplified in a suitable host cell. For example, if a humangene can cut in 2 places with the RE enzyme known as EcoR1, the desiredfragment with EcoR1 ends can be isolated and mixed with a plasmid thatwas also cut with EcoR1 in what is commonly known as a ligation mixture.Under the appropriate conditions in the ligation mixture, some of theisolated human gene fragments will match up with the ends of the plasmidmolecules. These newly joined ends can link together (ligated) toenzymatically recircularize the plasmid, now containing its new DNAinsert. The ligation mixture is then introduced into E. coli or anothersuitable host, and the newly engineered plasmids will be amplified asthe bacteria divide. In this manner, a relatively large number of copiesof the human gene may be obtained and harvested from the bacteria. Thesegene copies can then be further manipulated for the purpose of research,analysis, or production of its gene product protein.

Recombinant DNA technology is frequently embodied in the generation ofso-called “transgenes”. Transgenes frequently comprise a variety ofgenetic materials that are derived from one or more donor organisms andintroduced into a host organism. Typically, a transgene is constructedusing a cloning vector as the starting point or “backbone” of theproject, and a series of complex cloning steps are planned to assemblethe final product within that vector. Elements of a transgene,comprising nucleotide sequences, include, but are not limited to 1)regulatory promoter and/or enhancer elements, 2) a gene that will beexpressed as a mRNA molecule, 3) DNA elements that provide mRNA messagestabilization, 4) nucleotide sequences mimicking mammalian intronic generegions, and 5) signals for mRNA processing such as the poly-A tailadded to the end of naturally-occurring mRNAs. In some cases, anexperimental design may require addition of localization signal toprovide for transport of the gene product to a particular subcellularlocation.

Each of the elements of a transgene can be derived as a fragment of alarger DNA molecule that is cut from a donor genome, or, in some cases,synthesized in a laboratory. While the present invention employsendonucleases for the methods claimed herein, it is known that each ofthe smaller elements comprising, for example, the inserts or moduleswhich are used in the methods herein, can be created by de novosynthesis, recombineering, and/or PCR terminator overhang cloning. Onesuch method of synthesis of the component elements of a transgeneincludes the method disclosed by Jarrell et al. in U.S. Pat. No.6,358,712, which is incorporated herein by reference in its entirety.While Jarrell discloses a method for “welding” elements of a transgenetogether, only the methods of the present invention disclose a way to“unweld” and re-assemble the elements once they have been assembled.According to one aspect of the invention, each piece is assembled withthe others in a precise order and 5′-3′ orientation into a cloningvector plasmid.

The promoter of any gene may be isolated as a DNA fragment and placedwithin a synthetic molecule, such as a plasmid, to direct the expressionof a desired gene, assuming that the necessary conditions forstimulation of the promoter of interest can be provided. For example,the promoter sequences of the insulin gene may be isolated, placed in acloning vector plasmid along with a reporter gene, and used to study theconditions required for expression of the insulin gene in an appropriatecell type. Alternatively, the insulin gene promoter may be joined withthe protein coding-sequence of any gene of interest in a cloning vectorplasmid, and used to drive expression of the gene of interest ininsulin-expressing cells, assuming that all necessary elements arepresent within the DNA transgene so constructed.

A reporter gene is a particularly useful component of some types oftransgenes. A reporter gene comprises nucleotide sequences encoding aprotein that will be expressed under the direction of a particularpromoter of interest to which it is linked in a transgene, providing ameasurable biochemical response of the promoter activity. A reportergene is typically easy to detect or measure against the background ofendogenous cellular proteins. Commonly used reporter genes include butare not limited to LacZ, green fluorescent protein, and luciferase, andother reporter genes, many of which are well known to those skilled inthe art.

Introns, which are non-coding regions within mammalian genes, are notfound in bacterial genomes, but are required for proper formation ofmRNA molecules in mammalian cells. Therefore, any DNA construct for usein mammalian systems must have at least one intron. Introns may beisolated from any mammalian gene and inserted into a DNA construct,along with the appropriate splicing signals that allow mammalian cellsto excise the intron and splice the remaining mRNA ends together.

An mRNA stabilization element is a sequence of DNA that is recognized bybinding proteins that protect some mRNAs from degradation. Inclusion ofan mRNA stabilization element will frequently enhance the level of geneexpression from that mRNA in some mammalian cell types, and so can beuseful in some DNA constructs or transgenes. An mRNA stabilizationelement can be isolated from naturally occurring DNA or RNA, orsynthetically produced for inclusion in a DNA construct.

A localization signal is a sequence of DNA that encodes a protein signalfor subcellular routing of a protein of interest. For example, a nuclearlocalization signal will direct a protein to the nucleus; a plasmamembrane localization signal will direct it to the plasma membrane, etc.Thus, a localization signal may be incorporated into a DNA construct topromote the translocation of its protein product to the desiredsubcellular location.

A tag sequence may be encoded in a DNA construct so that the proteinproduct will have a unique region attached. This unique region serves asa protein tag that can distinguish it from its endogenous counterpart.Alternatively, it can serve as an identifier that may be detected by awide variety of techniques well known in the art, including, but notlimited to, RT-PCR, immunohistochemistry, or in situ hybridization.

With a complex transgene, or with one that includes particularly largeregions of DNA, there is an increased likelihood that there will bemultiple endonuclease recognition sites in these pieces of DNA. Recallthat the recognition sequences encoding any one hexanucleotide siteoccur every 4096 bp (4⁶). If a promoter sequence is 3000 bp and a geneof interest of 1500 bp are to be assembled into a cloning vector of 3000bp, it is statistically very likely that many sites of 6 or lessnucleotides will not be useful, since any usable sites must occur inonly two of the pieces. Furthermore, the sites must occur in theappropriate areas of the appropriate molecules that are to be assembled.In addition, most cloning projects will need to have additional DNAelements added, thereby increasing the complexity of the growingmolecule and the likelihood of inopportune repetition of any particularrestriction site. Since any restriction enzyme will cut at all of itssites in a molecule, if an endonuclease enzyme restriction sitereoccurs, all the inopportune sites will be cut along with the desiredsites, disrupting the integrity of the molecule. Thus, each cloning stepmust be carefully planned so as not to disrupt the growing molecule bycutting it with an endonuclease enzyme that has already been used toincorporate a preceding element. And finally, when a researcher wishesto introduce a completed transgene into a mammalian organism, thefully-assembled transgene construct frequently must be linearized at aunique recognition site at at least one end of the transgene, thusrequiring yet another unique recognition site found nowhere else in theconstruct. Since most DNA constructs are designed for a single purpose,little thought is given to any future modifications that might need tobe made, further increasing the difficulty for future experimentalchanges.

Traditionally, transgene design and construction consumes significantamounts of time and energy for several reasons, including the following:

1. There is a wide variety of endonuclease enzymes available that willgenerate an array of termini, however most of these are not compatiblewith each other. Many endonuclease enzymes, such as EcoR1, generate DNAfragments with protruding 5′ cohesive termini or “tails”; others (e.g.,Pst1) generate fragments with 3′ protruding tails, whereas still others(e.g., Bal1) cleave at the axis of symmetry to produce blunt-endedfragments. Some of these will be compatible with the termini formed bycleavage with other endonuclease enzymes, but the majority of usefulones will not. The termini that can be generated with each DNA fragmentisolation must be carefully considered in designing a DNA construct.

2. DNA fragments needed for assembly of a DNA construct or transgenemust first be isolated from their source genomes, placed into plasmidcloning vectors, and amplified to obtain useful quantities. The step canbe performed using any number of commercially-available or individuallyaltered cloning vectors. Each of the different commercially availablecloning vector plasmids were, for the most part, developedindependently, and thus contain different sequences and endonucleasesites for the DNA fragments of genes or genetic elements of interest.Genes must therefore be individually tailored to adapt to each of thesevectors as needed for any given set of experiments. The same DNAfragments frequently will need to be altered further for subsequentexperiments or cloning into other combinations for new DNA constructs ortransgenes. Since each DNA construct or transgene is custom made for aparticular application with no thought or knowledge of how it will beused next, it frequently must be “retro-fitted” for subsequentapplications.

3. In addition, the DNA sequence of any given gene or genetic elementvaries and can contain internal endonuclease sites that make itincompatible with currently available vectors, thereby complicatingmanipulation. This is especially true when assembling several DNAfragments into a single DNA construct or transgene.

Thus, there remains a need for a system that would allow the user torapidly assemble a number of DNA fragments into one molecule, despiteredundancy of endonuclease sites found at the ends and within the DNAfragments. Such a system might also provide a simple means for rapidlyaltering the ends of the fragments so that other endonuclease sequencesare added to them. Inclusion of single or opposing pairs of HE siteswould enhance the likelihood of having unique sites for cloning. Asystem that would also allow easy substitutions or removal of one ormore of the fragments would add a level of versatility not currentlyavailable to users. Therefore, a “modular” system, i.e. a systemallowing one to insert or remove DNA fragments or “inserts” into or outof “cassette” regions flanked by rare endonuclease sites within thecloning vector, would be especially useful and welcome to the field ofrecombinant DNA technology.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a method of rapidlyassembling DNA constructs or transgenes by using cloning vectorplasmids. The invention also provides a method that incorporatesmultiple DNA fragments, also known as both “inserts” or “modules”, suchas one each of a Promoter, Expression, and 3′ Regulatory nucleotidesequence, into a cloning vector plasmid in a single step, rather thanhaving to introduce each insert in a sequential manner. Such a method iscalled “Dynamic Vector Assembly” herein.

In one embodiment, the present invention provides a method forconstructing a transgene, comprising the steps of providing a cloningvector plasmid with a backbone able to accept a sequential arrangementof inserts, providing at least a first insert and a second insert to beincluded in the transgene, and transferring both the first insert andthe second insert into the backbone in a single reaction.

In another embodiment, the invention provides a method for making atransgene, comprising the steps of: providing a cloning vector plasmidcomprising first and second docking points; introducing first nucleotidesequences to be included in the transgene into a first shuttle vector;introducing second nucleotide sequences to be included in the transgeneinto a second shuttle vector; and transferring simultaneously the firstnucleotide sequences and the second nucleotide sequences from theshuttle vectors to the cloning vector plasmid, between the first andsecond docking points.

The invention also provides a method for making a transgene, comprisingthe steps of: providing a cloning vector plasmid comprising first andsecond docking points; introducing Promoter nucleotide sequences to beincluded in the transgene into a Promoter shuttle vector; introducingExpression nucleotide sequences to be included in the transgene into anExpression shuttle vector; introducing Regulatory nucleotide sequencesto be included in the transgene into a Regulatory shuttle vector; andtransferring simultaneously the Promoter, Expression and Regulatorynucleotide sequences from the Promoter, Expression and Regulatoryshuttle vectors to the cloning vector plasmid, between the first andsecond docking points.

In another embodiment, the invention provides a method forsimultaneously synthesizing an array of transgenes, comprising the stepsof: providing a primary cloning vector plasmid comprising a first and asecond docking point; introducing at least one Promoter nucleotidesequence to be included in the transgene into a corresponding Promotershuttle vector; introducing at least one Expression nucleotide sequenceto be included in the transgene into a corresponding Expression shuttlevector; introducing at least one Regulatory nucleotide sequence to beincluded in the transgene into a corresponding Regulatory shuttlevector; and transferring simultaneously the Promoter, Expression andRegulatory nucleotide sequences from the Promoter, Expression andRegulatory shuttle vectors to the cloning vector plasmid, between thefirst and second docking points, wherein at least two combinations ofone Promoter module, one Expression module, and one Regulatory moduleare transferred into two distinct primary cloning vector molecules.

In yet another embodiment, the invention provides a method for making amodular cloning vector plasmid for the synthesis of a transgene or othercomplicated DNA construct, the method comprising the steps of: providingthe cloning vector plasmid comprising a backbone, the backbonecomprising first and second docking points, each docking point beingfixed within the backbone and comprising at least one non-variable rareendonuclease site for an endonuclease enzyme; cleaving the first dockingpoint with a first endonuclease enzyme corresponding to the at least onenon-variable rare restriction site of the first docking point, leavingthe cleaved first docking point with a 3′ end; cleaving the seconddocking point with a second nuclease enzyme corresponding to the atleast one non-variable rare endonuclease site of the second dockingpoint, leaving the cleaved second docking point with a 5′ end; providingat least a first and a second insert, each insert comprising a 5′ end, anucleotide sequence of interest and a 3′ end, wherein the 5′ end of thefirst insert is compatible to the 3′ end of the cleaved first dockingpoint, the 3′ end of the second insert is compatible to the 5′ end ofthe cleaved second docking point, the 3′ end of the first insert beingcompatible to the 5′ end of the second insert to form a thirdnon-variable rare endonuclease site for a third endonuclease enzyme; andplacing the inserts and the cleaved cloning vector plasmid into anappropriate reaction mixture to cause simultaneous ligation andself-orientation of the first and second inserts between the first andsecond docking points within the backbone, re-forming the first andsecond docking points, and forming the modular cloning vector plasmid.

In another embodiment, the invention provides a method for synthesizinga transgene or other complicated DNA construct, comprising the steps of:providing a primary cloning vector plasmid comprising a backbone, thebackbone comprising at least a first docking point and a second dockingpoint, each docking point being fixed within the backbone and comprisingat least one rare restriction site for a non-variable rare restrictionenzyme; cleaving the first docking point with a first non-variable rarerestriction enzyme corresponding to one of the rare restriction sites ofthe first docking point, leaving the cleaved backbone with a 3′ end;cleaving the second docking point with a second non-variable rarerestriction enzyme corresponding to one of the restriction sites of thesecond docking point, leaving the cleaved backbone with a 5′ end,providing a Promoter insert into which a Promoter sequence of interest,a 5′ end that is compatible to the 3′ end of the first docking point,and a 3′ end; providing an Expression insert comprising an Expressionsequence of interest, a 5′ end that is compatible to the 3′ end of thePromoter insert to form a rare restriction site for a third non-variablerare restriction enzyme, and a 3′ end; providing a Regulatory insertcomprising a Regulatory sequence of interest, a 5′ end that iscompatible to the 3′ end of the Expression insert to form a rarerestriction site for a fourth non-variable rare restriction enzyme, anda 3′ end that is compatible to the 5′ end of the cleaved second dockingpoint which was cleaved in step ‘c’; and placing the Promoter,Expression and Regulatory inserts and the cleaved cloning vector plasmidinto an appropriate reaction mixture to cause simultaneous ligation,self-orientation and sequential placement of the Promoter, Expressionand Regulatory inserts between the first and second docking points,reforming the first and second docking points, and forming a modularprimary cloning vector plasmid.

In yet another embodiment, the invention provides a method forsimultaneously synthesizing an array of transgenes or other complicatedDNA constructs, comprising the steps of: providing at least one primarycloning vector plasmid comprising a backbone into which inserts having a5′ end, a nucleotide sequence of interest and a 3′ end can be inserted,the backbone operable to accept a sequential arrangement of Promoter,Expression, and Regulatory inserts and comprising at least a first and asecond docking point, each docking point being fixed within the backboneand comprising at least one restriction site for a non-variable rarerestriction enzyme; cleaving the first docking point with a firstnon-variable rare restriction enzyme corresponding to one of therestriction sites of the first docking point; cleaving the seconddocking point with a second non-variable rare restriction enzymecorresponding to one of the restriction sites of the second dockingpoint; providing at least one Promoter insert into which a Promoternucleotide sequence has been inserted, the 5′ end of the at least onePromoter insert compatible to the 3′ end of the first docking pointwhich was cleaved in step ‘b’; providing at least one Expression insertinto which an Expression nucleotide sequence has been inserted, the 5′end of the at least one Expression insert being compatible to the 3′ endof the at least one Promoter insert to form a restriction site for athird non-variable rare restriction enzyme; providing at least oneRegulatory insert into which a Regulatory nucleotide sequence has beeninserted, the 5′ end of the at least one Regulatory insert beingcompatible to the 3′ end of the at least one Expression insert to form arestriction site for a fourth non-variable rare restriction enzyme, the3′ end of the at least one Regulatory insert compatible to the 5′ end ofthe of the second docking point which was cleaved in step ‘c’; andthereafter placing at least two different types of at least one of thePromoter, Expression and Regulatory inserts, at least one of each of theremaining inserts, and the cleaved cloning vector plasmid into anappropriate reaction mixture to cause simultaneous ligation,self-orientation and sequential placement of one each of the Promoter,Expression and Regulatory inserts between the first and second dockingpoints within the backbone, thereby creating an array of plasmids havingdifferent combinations of Promoter, Expression and Regulatory insertswithin their backbone.

A further understanding of the nature and advantages of the presentinvention will be more fully appreciated with respect to the followingdrawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with a general description of the invention given above, andthe detailed description given below, serve to explain the principles ofthe invention.

FIG. 1 is a linear map of the module concept of the invention showing aP Shuttle vector that is insertable into a PE3 docking station, which isinsertable into a Primary docking station.

FIG. 2 is an illustration depicting assembly of a backbone vectorenabled by the relationships between restriction sites within shuttlevectors such as Promoter, Expression and 3′ Regulatory modules, and thedocking points on a primary cloning vector plasmid.

FIG. 3 is an illustration depicting assembled backbone vector of FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “chromatin modification domain” (CMD) refers tonucleotide sequences that interact with a variety of proteins associatedwith maintaining and/or altering chromatin structure.

As used herein, the term “cloning” refers to the process of ligating aDNA molecule into a plasmid and transferring it an appropriate host cellfor duplication during propagation of the host.

As used herein, the terms “cloning vector” and “cloning vector plasmid”are used interchangeably to refer to a circular DNA molecule minimallycontaining an Origin of Replication, a means for positive selection ofhost cells harboring the plasmid such as an antibiotic-resistance gene;and a multiple cloning site.

As used herein, the term “common” in relation to endonuclease sitesrefers to any endonuclease site that occurs relatively frequently withina genome.

As used herein, the phrase “compatible to” refers a terminus or end,either 5′ or 3′, of a strand of DNA which can form hydrogen bonds withany other complementary termini either cleaved with the same restrictionenzyme or created by some other method. Since any DNA that contains aspecific recognition sequence for a restriction enzyme will be cut inthe same manner as any other DNA containing the same sequence, thosecleaved ends will be complementary and thus compatible. Therefore, theends of any DNA molecules cut with the same restriction enzyme “match”each other in the way adjacent pieces of a jigsaw puzzle “match”, andcan be enzymatically linked together. Compatible ends will form arecognition site for a particular restriction enzyme when combinedtogether.

As used herein, the term “de novo synthesis” refers to the process ofsynthesizing double-stranded DNA molecules of any length by linkingcomplementary single-stranded DNA molecules compatible overhangs thatrepresent subsequences of the total desired DNA molecule.

As used herein, the term “DNA construct” refers to a DNA moleculesynthesized by consecutive cloning steps within a cloning vectorplasmid, and is commonly used to direct gene expression in anyappropriate cell host such as cultured cells in vitro, or a transgenicmouse in vivo. A transgene used to make such a mouse can also bereferred to as a DNA construct, especially during the period of timewhen the transgene is being designed and synthesized.

As used herein, the term “DNA fragment” refers to any isolated moleculeof DNA, including but not limited to a protein-coding sequence, reportergene, promoter, enhancer, intron, exon, poly-A tail, multiple cloningsite, nuclear localization signal, or mRNA stabilization signal, or anyother naturally occurring or synthetic DNA molecule, or any portionthereof. Alternatively, a DNA fragment may be completely of syntheticorigin, produced in vitro. Furthermore, a DNA fragment may comprise anycombination of isolated naturally occurring and/or synthetic fragments.

As used herein, the term “Docking Plasmid” refers to a specializedcloning vector plasmid used in the invention to assemble DNA fragmentsinto a DNA construct.

As used herein, the terms “endonuclease” or “endonuclease enzyme” refersto a member or members of a classification of catalytic molecules thatbind a recognition site encoded in a DNA molecule and cleave the DNAmolecule at a precise location within or near the sequence.

As used herein, the terms “endonuclease recognition site”, recognitionsite”, “cognate sequence” or “cognate sequences” refer to the minimalstring of nucleotides required for a restriction enzyme to bind andcleave a DNA molecule or gene.

As used herein, the term “enhancer region” refers to a nucleotidesequence that is not required for expression of a target gene, but willincrease the level of gene expression under appropriate conditions.

As used herein, the term “gene expression host selector gene” (GEH-S)refers to a genetic element that can confer to a host organism a traitthat can be selected, tracked, or detected by optical sensors, PCRamplification, biochemical assays, or by cell/organism survival assays(resistance or toxicity to cells or organisms when treated with anappropriate antibiotic or chemical).

As used herein, the terms “gene promoter” or “promoter” refer to anucleotide sequence required for expression of a gene, or any portion ofthe full-length promoter.

As used herein, the terms “insert” and “module” are essentiallyinterchangeable, with the only fine distinction being that an “insert”is inserted into the vector, and once it is inserted it is then morecommonly called a “module”. A module can then be removed from thevector. Also, the term insert is commonly used for an isolated moduleused as an insert into a modular acceptor vector.

As used herein, the term “intron” refers to the nucleotide sequences ofa non-protein-coding region within a mammalian cell gene found betweentwo protein-coding regions or exons.

As used herein, the term “localization signal” (LOC) refers tonucleotide sequences encoding a signal for subcellular routing of aprotein of interest.

As used herein, the term “multiple cloning site” (MCS) refers tonucleotide sequences comprising at least one unique endonuclease site,and, more typically, a grouping of unique endonuclease sites, for thepurpose of cloning DNA fragments into a cloning vector plasmid

As used herein, the term “mRNA stabilization element” refers a sequenceof DNA that is recognized by binding proteins thought to protect somemRNAs from degradation.

As used herein, the term “Origin of Replication” (ORI) refers tonucleotide sequences that direct replication or duplication of a plasmidwithin a host cell.

As used herein, the phrase “PCR terminator over-hang cloning technology”refers to the process of amplifying genetic modules using the polymerasechain reaction in conjunction with single-stranded DNA primers withprotected 5′ over-hanging nucleotides that can serve as junction siteswith complementary DNA over-hangs.

As used herein, the term “poly-A tail” refers to a sequence of adenine(A) nucleotides commonly found at the end of messenger RNA (mRNA)molecules. A Poly-A tail signal is incorporated into the 3′ ends of DNAconstructs or transgenes to facilitate expression of the gene ofinterest.

As used herein, the term “primer site” refers to nucleotide sequencesthat serve as DNA templates onto which single-stranded DNAoligonucleotides can anneal for the purpose of initiating DNAsequencing, PCR amplification, and/or RNA transcription.

As used herein, the term “pUC19” refers to a plasmid cloning vectorwell-known to those skilled in the art, and can be found in the NCBIGenbank database as Accession # L09137.

As used herein, the term “random nucleotide sequences” refers to anycombination of nucleotide sequences that do not duplicate sequencesencoding other elements specified as components of the same molecule.The number of nucleotides required in the random sequences is dependentupon the requirements of the endonuclease enzymes that flank the randomsequences. Most endonucleases require a minimum of 2-4 additional randomsequences to stabilize DNA binding. It is preferred that the number ofrandom sequences would be a multiple of 3, corresponding to the numberof nucleotides that make up a codon. The preferred minimum number ofrandom sequences is therefore 6, however, fewer or more nucleotides maybe used.

As used herein, the term “rare” in relation to endonuclease sites refersto an endonuclease site that occurs relatively infrequently within agenome.

As used herein, the term “recombination arm” refers to nucleotidesequences that facilitate the homologous recombination betweentransgenic DNA and genomic DNA. Successful recombination requires thepresence of a left recombination arm (LRA) and a right recombination arm(RRA) flanking a region of transgenic DNA to be incorporated into a hostgenome via homologous recombination.

As used herein, the term “recombineering” refers to the process of usingrandom or site-selective recombinase enzymes in conjunction with DNAsequences that can be acted on by recombinase enzymes to translocate aportion of genetic material from one DNA molecule to a different DNAmolecule.

As used herein, the term “reporter gene” refers to a nucleotidesequences encoding a protein useful for monitoring the activity of aparticular promoter of interest.

As used herein, the term “Shuttle Vector” refers to a specializedcloning vector plasmid used in the invention to make an intermediatemolecule that will modify the ends of a DNA fragment.

As used herein, the term “tag sequence” (TAG) refers to nucleotidesequences encoding a unique protein region that allows it to bedetected, or in some cases, distinguished from any endogenouscounterpart.

As used herein, the term “untranslated region” (UTR) refers tonucleotide sequences encompassing the non-protein-coding region of anmRNA molecule. These untranslated regions can reside at the 5′ end (5′UTR) or the 3′ end (3′ UTR) an mRNA molecule.

The present invention provides a method to take a newly manufacturedtransgene containing the modules and selectively remove one or more ofthe module and replace it with a different insert. This process iscalled herein “second pass” and “multiple threading”. The inventionfurther provides a method for creating an array of different transgenes,each having a different Promoter, Expression and Regulatory insert, byincorporating multiple different Promoter, Expression and Regulatoryinserts into a cloning vector plasmid in a single step. The presentinvention also provides a method that comprises the steps of providingcloning vector plasmids having newly introduced Promoter, Expression andRegulatory inserts combined together, removing the entire combination asa backbone vector, and inserting a multiple number of backbone vectorsinto a single cloning vector plasmid.

The present invention also provides a method to create a modular cloningvector plasmid for the synthesis of a transgene or other complicated DNAconstruct by providing a backbone having docking points therein. Eachdocking point represents an area in which there is preferably at leastone fixed non-variable rare endonuclease site, and more preferably fixedgroupings of two non-variable rare endonuclease sites, and mostpreferably fixed groupings of three non-variable rare endonucleasesites. A particular restriction site of each docking point is cleaved byits cognate endonuclease enzyme. This will create either a desired 5′ or3′ end which is compatible with the complementary 5′ or 3′ end of one ofthe pre-constructed inserts containing a nucleotide sequence of choice,such as a Promoter, Expression or Regulator nucleotide sequence. Atleast two inserts, each of which have 5′ and 3′ ends that are compatiblewith the cleaved docking point of interest, can be added along with thecleaved cloning vector plasmid to an appropriate reaction mixture, and,assuming the proper thermodynamic milieu, the inserts cansimultaneously, i.e. in a single step, become integrated into thecloning vector plasmid. During this singular addition and ligationreaction, the docking points are reformed and the cloning vector plasmidbecomes modular, in that the docking points and the connection betweenthe two modules can be re-cleaved with the appropriate restrictionenzymes. The module can then later be removed, and a new module can beput in its place.

One embodiment of the present invention relates to a method forconstructing a transgene, comprising the steps of providing a cloningvector plasmid with a backbone able to accept a sequential arrangementof inserts, providing at least a first insert and a second insert to beincluded in the transgene, and transferring both the first insert andthe second insert to the backbone in a single reaction. More preferablythe inserts consist of three inserts, specifically at least onePromoter, Expression, and Regulatory module.

Another embodiment of the invention is a method for making a transgenecomprising the steps of providing a cloning vector plasmid comprisingfirst and second docking points, introducing Promoter nucleotidesequences to be included in the transgene into a Promoter shuttlevector, introducing Expression nucleotide sequences to be included inthe transgene into an Expression shuttle vector, introducing Regulatorynucleotide sequences to be included in the transgene into a Regulatoryshuttle vector, transferring simultaneously the Promoter, Expression andRegulatory nucleotide sequences from the Promoter, Expression andRegulatory shuttle vectors to the cloning vector plasmid, between thefirst and second docking points.

It is preferred that both the 5′ and 3′ ends of each of the dockingpoints and each of the inserts all are compatible with a correspondingend of another docking point or insert. For example, if a first dockingpoint contains a restriction site for a non-variable rare restrictionenzyme such as SgrAI and that docking point is thereafter cleaved, thena first insert intended to be inserted at the 3′ end of the cleavedfirst docking point will contain a compatible 5′ end to create arestriction site for SgrAI when the insert is combined with the firstdocking point. A second docking point within the plasmid may, forexample, have a restriction site for a non-variable restriction enzymesuch as SwaI. Any second insert will have at its 3′ end a compatiblenucleotide sequence to combine with the cleaved 5′ end of the cleavedsecond docking point to create a restriction site for SwaI. Further, the3′ end of the first insert and the 5′ end of the second insert, in orderto simultaneously be inserted into the modular cloning vector plasmidand also thereafter be removed at the same point, must containcompatible ends to create a third restriction site for a thirdnon-variable rare restriction enzyme, such as PacI or SalI.

Sequential elements encoding the modular structure of the presentinvention can specifically comprise: three non-variable and uniquecommon restriction sites, a 5′ oligonucleotide primer site, a unique HEsite in a forward orientation, a pair of non-variable and unique, commonrestriction sites flanking random nucleotide sequences, a fixed groupingof non-variable rare restriction sites to define a 5′ portion of apromoter module, random nucleotide sequences, a fixed grouping ofnon-variable rare restriction sites that define a shared junctionbetween a 3′ position relative to the Promoter/intron module and a 5′position relative to an Expression module, random nucleotide sequences,a fixed grouping of non-variable rare restriction sites that define ajunction of a 3′ position relative to the Expression module and a 5′position relative to a 3′ Regulatory module, random nucleotidesequences, a fixed grouping of non-variable rare restriction sites thatdefine a 3′ position relative to a 3′ Regulatory module, a pair ofnon-variable and unique, common restriction sites flanking randomnucleotide sequences, a unique HE site in reverse orientation that isthe same HE site as that placed 3′ of the 5′ oligonucleotide primersite, a 3′ oligonucleotide primer site in reverse orientation, and fournon-variable and unique common restriction sites that define a 3′insertion site.

Other sequential elements encoding the modular structure of the presentinvention can specifically comprise: two non-variable and unique commonrestriction sites that define a 5′ insertion site, an oligonucleotideprimer site, a pair of unique HE sites in opposite orientation flankingrandom nucleotide sequences, a non-variable and unique, commonrestriction site that allows cloning of a shuttle vector moduledownstream of the pair of unique HE sites, a fixed grouping ofnon-variable rare restriction sites, random nucleotide sequences, afixed grouping of non-variable rare restriction sites, a unique HE sitein a forward orientation, a pair of non-variable and unique, commonrestriction sites flanking random nucleotide sequences, anoligonucleotide primer site, a pair of unique BstX I sites in oppositeorientations (wherein the variable nucleotide region in the BstX Irecognition site is defined by nucleotides identical to thenon-complementary tails generated by the ordering of two identical HErecognition sites arranged in reverse-complement orientation, a pair ofunique HE sites in opposite orientations flanking random nucleotidesequences; an oligonucleotide primer site in reverse-orientation, a pairof non-variable and unique, common restriction sites flanking randomnucleotide sequences, a unique HE site in reverse orientation, with theHE site being the same as the HE site in a forward orientation, a fixedgrouping of non-variable rare restriction sites, random nucleotidesequences, a fixed grouping of non-variable rare restriction sites, anon-variable and unique, common restriction site, a pair of unique HEsites in opposite orientation flanking random nucleotide sequences, anoligonucleotide primer site in reverse orientation, and threenon-variable and unique common restriction sites.

The present invention is a group of methods for assembling a variety ofDNA fragments into a de novo DNA construct or transgene by using cloningvectors optimized to reduce the amount of manipulation frequentlyneeded.

The primary vector, herein referred to as a Docking Plasmid, contains amultiple cloning site (MCS) with preferably 3 sets of rare endonucleasesites arranged in a linear pattern. This arrangement defines a modulararchitecture that allows the user to assemble multiple inserts into asingle transgene construct without disturbing the integrity of DNAelements already incorporated into the Docking Plasmid in previouscloning steps.

Two recognition sites for at least three HE are placed in oppositeorientation to flank three modular regions for the purpose of creating agene cassette acceptor site that cannot self-anneal. Because HE sitesare asymmetric and non-palindromic, it is possible to generatenon-complementary protruding 3′ cohesive tails by placing two HErecognition sites in opposite orientation. Thus, the HE I-SceI cuts itscognate recognition site as indicated by “/”:

5′ . . . TAGGGATAA/CAGGGTAAT . . . 3′, 3′. . . ATCCC/TATTGTCCCATTA . . . 5′.

The reverse placement of a second site within an MCS would generate twonon-complementary cohesive protruding tails:

5′ . . . TAGGGATAA    CCCTA . . . 3′ 3′ . . . ATCCCAATAGGGAT . . . 5′.

This is particularly useful when it is necessary to subclone largertransgenes into a vector. Due to the size of the insert, it isthermodynamically more favorable for a vector to self anneal rather thanaccept a large insert. The presence of non-complementary tails generatedby this placement of restriction sites provides chemical forces tocounteract the thermodynamic inclination for self-ligation.

The asymmetric nature of most HE protruding tails also creates apowerful cloning tool when used in combination with the BstX Iendonuclease enzyme site (5′ CCANNNNN/NTGG 3′, where ‘N’ can be anynucleotide). The sequence-neutral domain of BstX I can be used togenerate compatible cohesive ends for two reverse-oriented HE protrudingtails, while precluding self-annealing.

BstX I (I-Sce I Fwd.) I-Sce I Forward I-Sce I Reverse BstX I (I-Sce I Rev.)5′-CCAGATAA  CAGGGTAAT//ATTACCCTGTTAT     GTGG-3′3′-GGTC      TATTGTCCCATTA//TAATGGGAC     AATACACC-5′

Endonuclease sites used in the invention were chosen according to ahierarchy of occurrence. In order to determine the frequency ofendonuclease site occurrence, DNA sequence information corresponding tonineteen different genes was analyzed using Vector NTI software. Thissearch covered a total of 110,530 nucleotides of DNA sequence. Resultsfrom these analyses were calculated according to the number of instancesof an endonuclease site occurring within the analyzed 110,530nucleotides. Endonuclease sites were then assigned a hierarchicaldesignation according to four classifications, wherein “common” sitesoccur greater than 25 times per 110,530 nucleotides, “lower-frequency 6bp sites” occur between 6-24 times per 110,530 nucleotides, and “rare”sites occur between 0-5 times per 110,530 nucleotides. A partial list of“suitable” enzymes is hereby listed according to their occurrenceclassifications:

Common Endonuclease Enzymes:

Ase I, BamH I, Bgl II, Blp I, BstX I, EcoR I, Hinc II, Hind III, Nco I,Pst I, Sac I, Sac II, Sph I, Stu I, Xba I

Endonuclease enzymes that have a 6 bp recognition site, but have a lowerfrequency of occurrence:

Aar I, Aat II, AfI II, Age I, ApaL I, Avr II, BseA I, BspD I, BspE I,BstB I, Cla I, Eag I, Eco0109 I, EcoR V, Hpa I, Kpn I, Mfe I, Nar I, NdeI, NgoM IV, Nhe I, Nsi I, Pml I, SexA I, Sma I, Spe I, Xho I

Rare Endonuclease Enzymes:

Acl I, Asc I, AsiS I, BsiW I, Fse I, Mlu I, Not I, Nru I, Pac I, Pme I,Pvu I, Rsr II, Sal I, Sbf I, Sfi I, SgrA I, SnaB I, Swa I, PI-Sce I,I-Sce I, I-Ceu I, PI-Psp I, I-Ppo I, I-Tli I

Other endonucleases not included in these listings can also be used,maintaining the same functionality and the spirit and intent of theinvention.

The secondary vectors of the invention, herein known as Shuttle vectors,contain multiple cloning sites with common endonuclease sites flanked byrare endonuclease sites. The shuttle vectors are designed for cloningfragments of DNA into the common endonuclease sites between the raresites. The cloned fragments can subsequently be released by cleavage atthe rare endonuclease site or sites, and incorporated into the DockingPlasmid using the same rare endonuclease site or sites found in theshuttle vectors.

Thus, unlike conventional cloning vectors, the design of the MCS allows“cassettes” or modules of DNA fragments to be inserted into the modularregions of the Docking Plasmid. Likewise, each can be easily removedusing the same rare endonuclease enzymes, and replaced with any otherDNA fragment of interest. This feature allows the user to change thedirection of an experimental project quickly and easily without havingto rebuild the entire DNA construct. Thus, the cloning vector plasmidsof the present invention allow the user to clone a DNA fragment into anintermediate vector using common endonuclease sites, creating acassette-accepting module, and to then transfer that fragment to thedesired modular spot in the final construct by means of rareendonuclease sites. Furthermore, it allows future alterations to themolecule to replace individual modules in the Docking Plasmid with othercassette modules. The following descriptions highlight distinctions ofthe present invention compared with the prior art.

Individual components of a transgene (the promoter enhancer P, expressedprotein E, and/or 3′ regulatory region 3) can be assembled as modulestransferred from shuttle vectors into the PE3 Docking Station Plasmid.If higher orders of complexity are needed, the assembled transgenes, orother nucleotide sequences, can then be transferred into a PrimaryDocking Plasmid. Each of the five types of cloning vector plasmids willbe explained in greater detail to provide an understanding of thecomponents incorporated into each, beginning with the more complex PE3Docking Station Plasmid and the Primary Docking Plasmid.

The PE3 Docking Plasmid comprises a pUC19 backbone with the followingmodifications, wherein the sequences are numbered according to the pUC19Genbank sequence file, Accession # L09137:

1. Only sequences from 806 to 2617 (Afl3-Aat2) are used in the DockingPlasmid,

2. The BspH1 site at 1729 in pUC19 is mutated from TCATGA to GCATGA,

3. The Acl1 site at 1493 in pUC19 is mutated from AACGTT to AACGCT,

4. The Acl1 site at 1120 in pUC19 is mutated from AACGTT to CACGCT,

5. The Ahd1 site in pUC19 is mutated from GACNNNNNGTC to CACNNNNNGTC,

6. Sequences encoding BspH1/I-Ppo 1/BspH1 are inserted at the onlyremaining BspH1 site in pUC19 following the mutation step 2 in the listabove.

The multiple cloning site (MCS) in the PE3 Docking Plasmid comprises thefollowing sequential elements, in the order listed:

1. Three non-variable and unique common endonuclease sites that define a5′ insertion site for the mutated pUC19 vector described above (shownas, but not limited to, Aat II, Blp I, and Eco0109 I);

2. A T7 primer site;

3. A unique HE site (for example, 1-SceI (forward orientation));

4. A pair of non-variable and unique, common endonuclease sites flankingrandom nucleotide sequences that can serve as a chromatin modificationdomain acceptor module (RNAS-CMD-1) (for example, Kpn I and Avr II);

5. A fixed grouping of non-variable rare endonuclease sites that definethe 5′ portion of the promoter module (for example, AsiS I, Pac I, andSbf I);

6. Random nucleotide sequences that can serve as a Promoter/intronacceptor module (RNAS-P);

7. A fixed grouping of non-variable rare endonuclease sites that definethe shared junction between the 3′ portion of the Promoter/intron moduleand the 5′ portion of the Expression module (for example, SgrA I, AscI,and MluI);

8. Random nucleotide sequences that can serve as an expression acceptormodule (RNAS-E);

9. A fixed grouping of non-variable rare endonuclease sites that definethe junction of the 3′ portion of the Expression module and the 5′portion of the 3′ Regulatory module (for example, SnaB I, Not I, and SalI);

10. Random nucleotide sequences that can serve as a 3′ regulatory domainacceptor module (RNAS-3);

11. A fixed grouping of non-variable rare endonuclease sites that definethe 3′ portion of the 3′ Regulatory module (for example, Swa I, Rsr II,and BsiW I);

12. A pair of non-variable and unique, common endonuclease sitesflanking a random nucleotide sequence of DNA that can serve as achromatin modification domain acceptor module (RNAS-CMD-2) (for example,Xho I and Nhe I);

13. A unique HE site in reverse orientation that is identical to that initem 3, above;

14. A T3 primer site in reverse orientation; and

15. Four non-variable and unique common endonuclease sites that define a3′ insertion site for the mutated pUC19 vector described above (forexample, BspE I, Pme I, Sap I, and BspH I).

The Primary Docking Plasmid can be used to assemble two completedtransgenes that are first constructed in PE3 Docking Station Plasmids,or two homology arms needed to construct a gene-targeting transgene, orto introduce two types of positive or negative selection elements. Themultiple cloning site (MCS) in the Primary Docking Plasmid comprises thefollowing sequential elements, in the order listed:

1. Two non-variable and unique common endonuclease sites that define a5′ insertion site for the mutated pUC19 vector described above (forexample, Aat II and Blp 1);

2. An M13 Rev. primer site;

3. A pair of unique endonuclease flanking a random nucleotide sequenceof DNA that can serve as a genome expression host selector gene acceptormodule (RNAS-GEH-S1);

4. A non-variable and unique, common endonuclease site that allowscloning of a shuttle vector module downstream of the HE pair (forexample, EcoO109I);

5. A fixed grouping of non-variable rare endonuclease sites that definethe 5′ portion a Left Recombination Arm module (for example, AsiS I, PacI, and Sbf I);

6. Random nucleotide sequences that can serve as a Left RecombinationArm acceptor module (RNAS-LRA);

7. A fixed grouping of non-variable rare endonuclease sites that definethe 3′ portion of the Left Recombination Arm acceptor module (forexample, SgrA I, MluI, and AscI);

8. A unique HE site (for example, I-Ceu I (forward orientation));

9. A pair of non-variable and unique, common endonuclease sites flankinga random nucleotide sequence of DNA that can serve as a chromatinmodification domain acceptor module (RNAS-CMD-1) (for example, Kpn 1 andAvr II);

10. A T7 primer site;

11. A pair of unique BstX I sites in opposite orientation (wherein thevariable nucleotide region in the BstX I recognition site is defined bynucleotides identical to the non-complementary tails generated by theordering of two identical HE recognition sites arranged inreverse-complement orientation; for example, PI-SceI (forwardorientation) and PI-SceI (reverse orientation)) flanking a randomnucleotide sequence of DNA that can serve as a complex transgeneacceptor module (RNAS-PE3-1);

12. A pair of unique endonuclease sites flanking a random nucleotidesequence of DNA that can serve as a complex transgene acceptor module(RNAS-PE3-2);

13. A T3 primer site in reverse-orientation;

14. A pair of non-variable and unique, common endonuclease sitesflanking a random nucleotide sequence of DNA that can serve as achromatin modification domain acceptor module (RNAS-CMD-2) (for example,Xho I and Nhe I);

15. A unique HE site in reverse orientation that is identical to that initem 8 above;

16. A fixed grouping of non-variable rare endonuclease sites that definethe 5′ portion a Right Recombination Arm module (for example, SnaB I,Sal I, and Not I);

17. Random nucleotide sequences that can serve as a Right RecombinationArm acceptor module (RNAS-RRA);

18. A fixed grouping of non-variable rare endonuclease sites that definethe 3′ portion of the Right Recombination Arm acceptor module (forexample, Rsr II, Swa I, and BsiW I);

19. A non-variable and unique, common endonuclease site that allowscloning of a shuttle vector module (for example, BspE I);

20. A pair of unique endonuclease sites flanking a random nucleotidesequence of DNA that can serve as a genome expression host selector geneacceptor module (RNAS-GEH-S2);

21. An M13 Forward primer site placed in reverse orientation; and

22. Three non-variable and unique common endonuclease sites that definea 3′ insertion site for the mutated pUC19 vector described above (forexample, Pme I, Sap I, and BspH I).

Three cloning vector plasmids of the invention are known as ShuttleVectors. The Shuttle Vectors, like the PE3 and Primary Docking Plasmids,are also constructed from a pUC19 backbone. Just like the PE3 andPrimary Docking Plasmids, each Shuttle Vector has the same modificationsto the pUC19 backbone listed as 1 through 6, above. The individualShuttle Vectors (SV) are identified as Shuttle Vector Promoter/intron(P), Shuttle Vector Expression (E), and Shuttle Vector 3′Regulatory (3);henceforth SVP, SVE, and SV3, respectively. Each is described more fullybelow.

Shuttle Vector P (SVP):

SVP is a cloning vector plasmid that can be used to prepare promoter andintron sequences for assembly into a transgene construct. An example ofan SVP Plasmid can comprise the following sequential elements in theMCS, in the order listed:

1. Two non-variable and unique, common endonuclease sites that define a5′ insertion site for the mutated pUC19 vector described above (forexample, AatII and BlpI);

2. A T7 primer site;

3. A non-variable and unique, common endonuclease site that allowsefficient cloning of a shuttle vector module downstream of the T7 primersite (for example, Eco0109I);

4. A fixed grouping of non-variable rare endonuclease sites that definethe 5′ portion of the promoter module (for example, AsiSI, Pac I, andSbf I). These non-variable rare endonuclease sites provide the dockingpoint represented by the star at the 5′ end of the Promoter Vector ofFIG. 2;

5. A variable MCS comprising any grouping of common or rare endonucleasesites that are unique to the shuttle vector;

6. A fixed grouping of non-variable rare endonuclease sites that definethe 3′ portion of the promoter module (for example, SgrA I, AscI, andMluI). These non-variable rare endonuclease sites provide the dockingpoint represented by the circle at the 3′ end of the Promoter Vector ofFIG. 2;

7. A non-variable and unique, common endonuclease site that allowsefficient cloning of a shuttle vector module upstream of the T3 primersite (for example, BspEI);

8. A reverse-orientation T3 primer site; and

9. Two non-variable and unique, common endonuclease sites that define a3′ insertion site for the mutated pUC19 vector described above (forexample, PmeI and SapI).

Shuttle Vector E (SVE):

This is a cloning vector plasmid that can be used to prepare sequencesto be expressed by the transgene for assembly into a transgeneconstruct. An example of an SVE plasmid can comprise the followingsequential elements in the MCS, in the order listed:

1. Two non-variable and unique, common endonuclease sites that define a5′ insertion site for the mutated pUC19 vector described above (forexample, AatII and BIp \I);

2. A T7 primer site;

3. A non-variable and unique, common endonuclease site that allowsefficient cloning of a shuttle vector module downstream of the T7 primersite (for example, Eco0109\l);

4. A fixed grouping of non-variable rare endonuclease sites that definethe 5′ portion of the expression module (for example, SgrA I, AscI, andMluI). These non-variable rare endonuclease sites provide the dockingpoint represented by the circle at the 5′ end of the Expression Vectorof FIG. 2;

5. A variable MCS consisting of any grouping of common or rareendonuclease sites that are unique to the shuttle vector;

6. A fixed grouping of non-variable rare endonuclease sites that definethe 3′ portion of the expression module (for example, SnaBI, NotI, andSalI). These non-variable rare endonuclease sites provide the dockingpoint represented by the triangle at the 3′ end of the Expression Vectorof FIG. 2;

7. A non-variable and unique, common endonuclease site that allowsefficient cloning of a shuttle vector module upstream of the T3 primersite (for example, BspEI);

8. A reverse-orientation T3 primer site; and

9. Two non-variable and unique, common restriction sites that define a3′ insertion site for the mutated pUC19 vector described above (forexample, PmeI and SapI).

Shuttle Vector 3 (SV3):

This is a cloning vector plasmid that can be used to prepare 3′regulatory sequences for assembly into a transgene construct. An exampleof an SV3 plasmid can comprise the following elements in the MCS, in theorder listed:

1. Two non-variable and unique, common endonuclease sites that define a5′ insertion site for the mutated pUC19 vector described above (forexample, AatII and BlpI);

2. A T7 primer site;

3. A non-variable and unique, common endonuclease site that allowsefficient cloning of a shuttle vector module downstream of the T7 primer(for example, Eco0109I);

4. A fixed grouping of non-variable rare endonuclease sites that definethe 5′ portion of the 3′ regulatory module (for example, SnaBI, NotI,and SalI). These non-variable rare endonuclease sites provide thedocking point represented by the triangle at the 5′ end of theRegulatory Vector of FIG. 2.

5. A variable MCS consisting of any grouping of common or rareendonuclease sites that are unique to the shuttle vector;

6. A fixed grouping of non-variable rare endonuclease sites that definethe 3′ portion of the 3′ regulatory module (for example, SwaI, RsrII,and BsiWI). These non-variable rare endonuclease sites provide thedocking point represented by the square at the 3′ end of the RegulatoryVector of FIG. 2;

7. A non-variable and unique, non-rare endonuclease site that allowsefficient cloning of a shuttle vector module upstream of the T3 primersite (for example, BspEI);

8. A reverse-orientation T3 primer site; and

9. Two non-variable and unique, non-rare endonuclease sites that definea 3′ insertion site for the mutated pUC19 vector described above (forexample, PmeI and SapI).

While the present invention discloses methods for building transgenes inplasmid cloning vectors, similar methods can be used to build transgenesin larger extrachromosomal DNA molecules such as cosmids or artificialchromosomes, including bacterial artificial chromosomes (BAC). For usein plants, a T1 vector may also be used. The wide variety of geneticelements that can be incorporated into the plasmid cloning vectors alsoallow transfer of the final transgene products into a wide variety ofhost organisms with little or no further manipulation.

FIGS. 2 and 3 are a general illustration of the modularity of theinvention. As shown in FIG. 2, there is one each of a Promoter,Expression, and 3′ Regulatory shuttle vector. Flanking each insertwithin the shuttle vectors are endonuclease restriction sites that arespecific for creating a docking point. More specifically, in FIG. 2, thePromoter insert (P) is flanked by a first group of one or moreendonuclease restriction sites represented by astar at the 5′ end and asecond group of one or more endonuclease restriction sites representedby a circle at the 3′ end; the Expression module is flanked by thesecond group of endonuclease restriction sites represented by the circleat the 5′ end and a third group of one or more endonuclease restrictionsites represented by a triangle at the 3′ end; and the 3′ Regulatorymodule (3) is flanked by the third group of endonuclease restrictionsites represented by the triangle at the 5′ end and a fourth group ofone or more endonuclease restriction sites represented by a square atthe 3′ end. Cleaving each endonuclease recognition site by theendonuclease specific for that site creates sticky ends at the 5′ and 3′end of each module, as indicated in bottom portion of FIG. 2 by theinserts at the end of the dashed line arrows. The modules can now becombined with a cloning vector plasmid which has also been cleaved atits two fixed docking points by endonucleases specific for the firstgroup of endonuclease restriction sites (represented by the star) andthe fourth group of endonuclease restriction sites (represented by thesquare). When the modular vectors are placed with the cloning vectorplasmid in an appropriate reaction mixture, the cleaved sticky ends(represented by hollow stars, circles, triangles and squares) of eachmodular vector will self-orient within the plasmid and sequentiallyligate, with the cleaved star ends combining, the cleaved circle endscombining, the cleaved triangle ends combining, and the cleaved squareends combining. This results in an assembled backbone vector shown inFIG. 3. Further, each of the combined groups of endonuclease sitesrepresented by the star, circle, triangle, and square can once again becleaved by its corresponding specific endonuclease, such that aparticular insert can later be removed and replaced with another insertof interest.

Multiple backbone vectors (example, PE3-1 and PE3-2) can be insertedinto a single docking plasmid. The asymmetric nature of the protrudingtails of an endonuclease such as I-Sce I, as with other HE's, creates apowerful cloning tool when used in combination with the BstX Iendonuclease enzyme site (5′ CCANNNNN/NTGG 3′, where ‘N’ can be anynucleotide). The sequence-neutral domain of BstX I can be used togenerate compatible cohesive ends for two reverse-oriented I-Sce Iprotruding tails, while precluding self-annealing. With this method, afirst insert, PE3-1, having an I-Sce-1 site at its ends can be placed ina cloning vector plasmid by cleaving the plasmid at the Bstx1/SceIendonuclease sites. One can then cut again with I-SceI and insert aPE3-2 having I-Sce-1 site at its ends. This entire backbone can then becleaved from its docking plasmid by PI-Sce I and inserted into anotherdocking plasmid that contains BstX I/PI-Sce I endonuclease sites. Thissecond docking plasmid also has endonuclease sites for PI-Sce I, intowhich yet another module for a separate docking plasmid, possiblecontaining a PE3-3 and PE3-4, can be inserted (not shown). In thismanner, a researcher can get more information into one cell, that is,one can insert multiple genes within the context of a single vector,which has not previously been accomplished by those skilled in the art.Such a novel process can save a both money and time for researchersworking in this field.

EXAMPLES Example 1 PE3 Docking Plasmid

As an example of the method of practicing the present invention, atransgene can be constructed containing these elements:

1. Nucleotide sequences of the human promoter for surfactant protein C(SP-C);

2. Sequences encoding the protein product of the mouse genegranulocyte-macrophage colony-stimulating factor-receptor beta c(GMRβc);

3. Rabbit betaglobin intron sequences; and

4. SV40 poly-A signal.

The SP-C sequences contain internal BamH1 sites, and can be releasedfrom its parental plasmid only with Not1 and EcoR1. GMRβc has aninternal Not1 site, and can be cut from its parental plasmid with BamH1and Xho1. The rabbit betaglobin intron sequences can be cut out of itsparental plasmid with EcoR1. The SV-40 poly-A tail can be cut from itsparental plasmid with Xho1 and Sac1. Because of redundancy of several ofendonuclease sites, none of the parental plasmids can be used toassemble all the needed fragments.

The steps used to build the desired transgene in the PE3 Docking Plasmidinvention are as follows.

1. Since Not1 and PspOM1 generate compatible cohesive ends, the humanSP-C promoter sequences are excised with Not1 and EcoR1 and cloned intothe PspOM1 and EcoR1 sites of Shuttle Vector P. The product of thisreaction is called pSVP-SPC

2. Following propagation and recovery steps well known to those skilledin the art, the rabbit betaglobin intron sequences are cloned into theEcoR1 site of pSVP-SPC. Orientation of the intron in the resultantintermediate construct is verified by sequencing the product, calledpSVP-SPC-rβG.

3. The promoter and intron are excised and isolated as one contiguousfragment from pSVP-SPC-rβG using AsiS1 and Asc1. Concurrently, the PE3Docking Plasmid is cut with AsiS1 and Asc1 in preparation for ligationwith the promoter/intron segment. The promoter/intron fragment isligated into the Docking Plasmid, propagated, and recovered.

4. The Xho1 site of the GMRβc fragment is filled in to create a blunt 3′end, using techniques well known to those skilled in the art. It is thencloned into the BamH1 site and the blunt-ended Pvu2 site ofpSVP-SPC-rβG. The resultant plasmid (pDP-SPC-GMRβc-rβG) was propagatedand recovered.

5. The final cloning step is the addition of the SV-40 Poly-A tail. TheSV40-polyA fragment is cut out with Xho1 and Sac1, as is the recipientvector pDS1-SPC-GMRβc-rbβG. Both pieces of DNA are gel purified andrecovered. A ligation mix is prepared with a 10:1 molar ratio ofSV-40polyA to pDS1-SPC-GMRβc-rβG. The ligation products are propagatedand harvested. The new plasmid, pDS1-SPC-GMRββc-rβG-pA contains allelements required for the transgene, including a unique endonucleasesite at the 3′ end with which the entire pDS1-SPC-GMRβc-rβG-pA plasmidcan be linearized for transfection into eukaryotic cells ormicroinjection into the pronucleus of a fertilized ovum.

Example 2 Dynamic Vector Assembly

Dynamic Vector Assembly is illustrated in the following example:

1. Promoter sequences from the human cytomegalovirus (CMV) are insertedinto a P Shuttle Vector (SVP), having AsiSI and Ase I endonuclease atthe 5′ and 3′ portions, respectively. Plasmids are amplified, and thepromoter module is cleaved from the vector by AsiS I and Asc Iendonuclease digestion and isolated.

2. Sequences encoding a luciferase protein are inserted into anExpression Shuttle Vector (SVE), having Asc I and Not I endonuclease atthe 5′ and 3′ portions, respectively. Plasmids are amplified, and theExpression module is cleaved from the vector by Asc I and Not Iendonuclease digestion and isolated.

3. Sequences encoding a mammalian intron and SV40 poly-adenylation siteare inserted into a 3′ Regulatory Shuttle vector (SV3), having Not I andBsiW I endonuclease at the 5′ and 3′ portions, respectively. Plasmidsare amplified, and the Regulatory module is cleaved from the vector byNot I and BsiW I endonuclease digestion and isolated.

4. The endonuclease recognition sites in a Docking Vector plasmid havingAsiS I and BsiW I endonuclease at the 5′ portion of the promoter moduleand the 3′ portion of the regulator module, respectively, are cleavedwith AsiS I and BsiW I endonuclease and isolated.

5. The Promoter, Expression, and Regulatory modules are combined withthe Docking Vector Plasmid in a ligation mixture. Following anincubation of 2 hours, the ligation mixture is used to transform E.coli, which are then spread on an LB agar plate with ampicillin. Theplate is incubated at 37° C. overnight. Colonies are isolated andpropagated in individual liquid LB broth cultures. The plasmid DNA isisolated from each LB broth culture. The DNA is analyzed by endonucleasemapping to determine whether the plasmids from each colony contain thethree modular inserts (Promoter, Expression and Regulatory). A plasmidthat contains the three modular inserts is identified as the transgenepCMV-luc-SV40 pA. It can be linearized using I-Sce I endonuclease andinjected into mouse pronuclei to generate CMV-luciferase mice. The CMVpromoter in this example directs the expression of the luciferase genein all tissues of a host organism, such as a CMV-luciferase mouse.

Example 3 Redesign of a Dynamic Vector Assembly

If the researcher now wishes to refine the expression pattern so thatluciferase is expressed only in a particular tissue or cell-type, he orshe can quickly and easily replace the CMV promoter with one that willprovide a restricted expression pattern. The following exampleillustrates the use of the invention to facilitate rapid redesign ofpCMV-luc-pA:

1. A neuron-specific promoter, Neuron-Specific Enolase (NSE), isinserted into a P Shuttle Vector (SVP) and prepared as the PromoterModule in the previous example.

2. pCMV-luc-pA is cleaved with AsiS I and Asc I to remove the CMVPromoter Module. The remainder of the Docking Vector Plasmid containingintact Expression and Regulatory Modules is isolated.

3. The NSE Promoter Module is placed in a ligation mixture with theremainder of the Docking Vector Plasmid containing intact Expression andRegulatory Modules. Following incubation for 2 hours, the new ligationmixture is used to transform E. coli. The E. coli mixture is spread onan LB agar plate with ampicillin, as in the previous example. Coloniesare isolated the following day, propagated, and plasmid DNA is isolatedfrom each. Endonuclease mapping is used to identify plasmids thatcontain the desired NSE Promoter module.

Example 4 Array of Transgenes

The following example is an illustration of the use of the invention torapidly assemble an array of transgenes, each containing a differentcombination of Promoter, Expression, and Regulatory modules. A series ofsix shuttle vectors and a PE3 docking station vector will be used togenerate eight different vector products using combinatorial assembly.The series of six shuttle vectors consists of two P-Shuttles (SVP), twoE-Shuttles (SVE), and two 3-Shuttles (SV3). The two discrete P-Shuttles(SVP) contain either a human cytomegalovirus (CMV) promoter or a mouseSPC lung-specific promoter, and each has AsiS I and Asc I endonucleaseat the 5′ and 3′ portions, respectively. The two discrete E-Shuttlescontain either a Luciferase cDNA or an EGFP cDNA, and each has Asc I andNot I endonuclease at the 5′ and 3′ portions, respectively. The twodiscrete 3-Shuttle vectors contain either an SV40 polyA signal or the 3′regulatory region of the human growth hormone (hGH), and each has Not Iand BsiW I endonuclease at the 5′ and 3′ portions, respectively.

The promoter modules are released from their respective SVP shuttlevectors by individually digesting appropriate shuttle vector with theAsiS I and the Asc I endonucleases. The resulting restriction productsare individually subjected to gel electrophoresis and the DNA bandcorresponding to the appropriate promoter module is subjected to gelpurification. This procedure will yield either a CMV promoter module oran SPC promoter module bounded on the 5′ side by an AsiS I overhang andby an Asc I overhang on the 3′ end.

The expression modules are released from their respective SVE shuttlevectors by individually digesting appropriate shuttle vector with theAsc I and the Not I restriction endonucleases. The resulting restrictionproducts are individually subjected to gel electrophoresis and the DNAband corresponding to the appropriate expression module is subjected togel purification. This procedure will yield either a Luciferaseexpression module or an EGFP expression module bounded on the 5′ side byan Asc I overhang and by a Not I overhang on the 3′ end.

The 3′ regulatory modules are released from their respective SV3 shuttlevectors by individually digesting appropriate shuttle vector with theNot I and the BsiW I restriction endonucleases. The resultingrestriction products are individually subjected to gel electrophoresisand the DNA band corresponding to the appropriate 3′ regulatory moduleis subjected to gel purification. This procedure will yield either aSV40 3′ regulatory module or an hGH 3′ regulatory module bounded on the5′ side by a Not I overhang and by a BsiW I overhang on the 3′ end.

The PE3 docking station vector is prepared by digesting with the AsiS Iand the BsiW I restriction endonucleases. To help prevent future vectorre-ligation, the vector restriction digest is exposed to calf intestinalphosphatase (CIP) for one hour at 37° C. The resulting CIP-treatedvector restriction product is then subjected to gel electrophoresis andthe DNA band corresponding to linearized PE3 vector backbone issubjected to gel purification.

Samples from the seven resulting gel-purified DNA fragments are analyzedfor identity, integrity, purity, and quantity by running out on adiagnostic electrophoretic gel. Quantitative data concerning therelative abundance of the purified PE3 docking station vector and therespective DNA modules is used to define the amount of each componentneeded for a combinatorial ligation reaction.

When setting up a ligation reaction, there are two strategies thatfrequently lead to successful results. The first strategy is to set upligation reaction mixtures wherein the insert-to-vector ratio is about3:1. The second strategy, used when more than one insert is beingintroduced to a single vector simultaneously, is to introduce a molarequivalent of each genetic module that will be inserted into the vector.This can be achieved either by adding a variable volume of the modulesto a reaction container in order to obtain molar equivalence in thecontext of the ligation reaction mixture, or by adding a neutral buffersolution to each of the purified modules so that their concentrationsare equivalent on a molar ratio basis. In this example, the gel-purifiedvector and insert fragments have all been adjusted to molar equivalenceusing the buffer 10 mM Tris, pH 8.0. The total ligation reaction volumeis set at 150 microliters. The ligation reaction mixture consists of thefollowing constituents: 39 microliters of ultrapure water, 15microliters of 10× Ligase buffer, 5 microliters of the purified PE3vector backbone, 15 microliters of the purified CMV Promoter module, 15microliters of the purified SPC Promoter module, 15 microliters of thepurified Luciferase expression module, 15 microliters of the purifiedEGFP expression module, 15 microliters of the purified SV40 3′regulatory module, 15 microliters of the purified hGH 3′ regulatorymodule, and 1 microliter of ligase enzyme. The resulting reactioncomponents are thoroughly mixed and then incubated overnight at 16° C.

The predicted vector ligation products include the following:

-   pCMV-EGFP-SV40-   pCMV-EGFP-hGH-   pCMV-Luciferase-SV40-   pCMV-Luciferase-hGH-   pSPC-EGFP-SV40-   pSPC-EGFP-hGH-   pSPC-Luciferase-SV40-   pSPC-Luciferase-hGH

The ligation mixture is then used to transform E. coli, which are thenspread on an LB agar plate with ampicillin. The plate is incubated at37° C. overnight. Colonies are isolated and propagated in individualliquid LB broth cultures. The plasmid DNA is isolated from each LB brothculture. The DNA is analyzed by endonuclease mapping to determine theidentity of the resulting vector incorporated into each colony.

In the preceding example, one of the predicted vector products(pCMV-EGFP-SV40) was not produced during the first combinatorialprocess. One vector that was successfully produced(pCMV-Luciferase-SV40) can, however, serve as a vector backbone forproducing the desired pCMV-EGFP-SV40 vector. This technique can bereferred to as “Second Pass Assembly”.

Example 5 Second Pass Assembly

In order to build the desired pCMV-EGFP-SV40 vector, thepCMV-Luciferase-SV40 vector product of Example 4 is digested with Asc Iand Not I, CIP-treated, and subsequently gel-purified. This linearizedvector fragment, in which the Luciferase module has been removed, isincubated in a ligation mixture containing the EGFP module produced inthe previous example of combinatorial vector assembly.

The ligation mixture is used to transform E. coli, which are then spreadon an LB agar plate with ampicillin. The plate is incubated at 37° C.overnight. Colonies are isolated and propagated in individual liquid LBbroth cultures. The plasmid DNA is isolated from each LB broth culture.The DNA is analyzed by endonuclease mapping to determine whether theplasmids from each colony contain the EGFP insert.

Among the many advantages of the present invention, it can readily beappreciated that one can rapidly assemble an array of transgenes, eachcontaining a different combination of Promoter, Expression, andRegulatory modules, in a very short period of time, as well as quicklyand easily vary or redesign a newly assemble transgene. In the past,varying an assembled transgene using known methods to create an array ofdifferent transgenes, each having different Promoter, Expression, andRegulatory modules would usually take a year or more of laboratory time.Using the methods of the present invention, one can make the same numberof desired transgenes within days or weeks, and then do the desiredtesting of each, thereby saving the researcher a previously large amountof time. Further, both Dynamic Vector Assembly, in which one each of aPromoter, Expression and Regulatory insert can be inserted into a singlebackbone at the same time, and the combination method described, inwhich two P-Shuttles, two E-Shuttles, and two Regulatory-Shuttles areall combined to create eight different types of transgenes, can be usedto save precious time and money for researchers. Shuttles that wereoriginally created by de novo synthesis, recombineering, and PCRterminator over-hang cloning methods can be taken and used with thedocking point technology of the present invention to rapidly assemblethese pre-made elements into a multitude of transgenes.

While the present invention has been illustrated by the description ofembodiments thereof, and while the embodiments have been described indetail, it is not intended to restrict or in any way limit the scope ofthe appended claims to such detail. Additional advantages andmodifications will be readily apparent to those skilled in the art. Theinvention in its broader aspects is therefore not limited to thespecific details, representative methods and structures, and illustratedexamples shown and described. Accordingly, departures may be made fromsuch details without departing from the scope or spirit of Applicant'sgeneral inventive concept.

What is claimed is:
 1. A method for simultaneously synthesizing an arrayof transgenes, comprising: a. providing a primary cloning vector plasmidcomprising a first and a second docking point, wherein each dockingpoint comprises at least one endonuclease site of greater than sixnucleotides, and wherein the cloning vector plasmid further comprises aunique homing endonuclease site in a forward orientation locatedupstream from the 5′ end of the first docking point and a unique homingendonuclease site in a reverse orientation located downstream from the3′ end of the second docking point; b. introducing at least one Promoternucleotide sequence into a corresponding Promoter shuttle vector; c.introducing at least one Expression nucleotide sequence into acorresponding Expression shuttle vector; d. introducing at least oneRegulatory nucleotide sequence into a corresponding Regulatory shuttlevector; e. simultaneously ligating the Promoter, Expression, andRegulatory nucleotide sequences from the Promoter, Expression, andRegulatory shuttle vectors to the cloning vector plasmid, between thefirst and second docking points, thereby forming a transgene constructcomprising the Promoter, Expression, and Regulatory nucleotidesequences; f. digesting the cloning vector plasmid with homingendonucleases that recognize the unique homing endonuclease sites in(a), thereby releasing the transgene construct; g. providing a secondcloning vector plasmid comprising the same unique homing endonucleasesite in a forward orientation and the same unique homing endonucleasesite in a reverse orientation as the first cloning vector plasmid; h.digesting the second cloning vector plasmid with the homing endonucleaseused in (f); and i. ligating the transgene construct of (f) into thesecond cloning vector plasmid.
 2. The method of claim 1, wherein theinserts are created by a method selected from the group consisting of denovo synthesis, recombineering, and PCR terminator over-hang cloning. 3.The method of claim 1, further comprising ligating a second transgeneconstruct into the second cloning vector plasmid.
 4. The method of claim1, wherein the unique homing endonuclease site in the forwardorientation is the same as the unique homing endonuclease site in thereverse orientation.
 5. The method of claim 1, wherein the ratio of theprimary cloning vector plasmid to each shuttle vector is about 3:1.